Traditionally, activity assays of polymerases, including DNA-directed DNA polymerases, DNA-directed RNA polymerases, RNA-directed DNA polymerases and RNA-directed RNA polymerases, involve a primed nucleic acid template, 32P-labeled dNTPs, a polymerase and a buffer system. The quantity of the incorporated radio-isotope labeled dNTPs into the final product is used to measure the activity of the enzyme. This assay method requires skilled experts, specialized facilities for radioisotopes and produces radioisotope-contaminated waste.
Non-radioactive methods that are now available, including the M13/SYBR Green assay, and a real-time assay using a DNA binding dye, DAPI, have the disadvantage of being complicated, involving expensive instruments for monitoring the reactions, lengthy preparation time and a large quantity of reagents.
Therefore, in view of the state of the art, a need exists for broadly applicable assays for the presence or activity of polymerases using a non-radioactive method.
Fluorescence resonance energy transfer (FRET) is a form of molecular energy transfer (MET), a process by which energy is passed non-radioactively between a donor molecule and an acceptor molecule. FRET arises from the properties of certain chemical compounds; when excited by exposure to particular wavelengths of light, they emit light (i.e., they fluoresce) at a different wavelength. Such compounds are termed fluorophores or fluorescent labels. In FRET, energy is passed non-radioactively over a long distance (e.g., 10-100 Angstroms) between a donor molecule, which may be a fluorophore, and an acceptor molecule, which may be a quencher or another fluorophore. The donor absorbs a photon and transfers this energy non-radioactively to the acceptor (Forster, 1949, Z. Naturforsch. A4:321-327; Clegg, 1992, Methods Enzymol. 211:353-388).
When two fluorophores whose excitation and emission spectra overlap are in close proximity, excitation of one fluorophore will cause it to emit light at wavelengths that are absorbed by, and that stimulate, the second fluorophore, causing it in turn to fluoresce. In other words, the excited-state energy of the first (donor) fluorophore is transferred by a resonance induced dipole-dipole interaction to the neighboring second (acceptor) fluorophore. As a result, the lifetime of the donor molecule is decreased and its fluorescence is quenched, while the fluorescence intensity of the acceptor molecule is enhanced and depolarized. When the excited-state energy of the donor is transferred to a non-fluorophore acceptor, the fluorescence of the donor is quenched without subsequent emission of fluorescence by the acceptor. In this case, the acceptor functions as a quencher.
Pairs of molecules that can engage in fluorescence resonance energy transfer (FRET) are termed FRET pairs. In order for energy transfer to occur, the donor and acceptor molecules must typically be in close proximity (e.g., up to 70 to 100 Angstroms) (Clegg, 1992, Methods Enzymol. 211:353-388; Selvin, 1995, Methods Enzymol. 246:300-334). The efficiency of energy transfer falls off rapidly with increased distance between the donor and acceptor molecules. Effectively, this means that FRET can most efficiently occur up to distances of about 70 Angstroms.
The subject matter discussed in the background section should not be assumed to be prior art merely as a result of its mention in the background section. Similarly, a problem mentioned in the background section or associated with the subject matter of the background section should not be assumed to have been previously recognized in the prior art. The subject matter in the background section merely represents different approaches.